The long-term objective of this application is to understand the molecular bases that regulate the determination, differentiation and maintenance of different retinal cell types. A number of transcription factors have been shown to play a key role during vertebrate retinogenesis. Among them is the POU domain transcription factor Brn3b which is critically required for the development of retinal ganglion cells. The Barhl2 homeodomain transcription factor may also play an important role in retinal development as it is expressed in ganglion cells as well as in neurons within the inner nuclear layer of the retina. [unreadable] [unreadable] The studies outlined in this application are designed to provide integrated approaches to address the fundamental mechanisms governing vertebrate retinal development, using the Brn3b and Barhl2 transcription factors as models for our analyses. Three specific aims will be pursued: i) to investigate the mechanisms by which Bm3b controls the differentiation and survival of retinal ganglion cells. These studies aim to identify Brn3b downstream genes by a "candidate gene" approach as well as the microarray technology followed by tests of their biological significance; ii) to identify and functionally characterize proteins interacting with Brn3b. We aim to identify Brn3b binding proteins that may regulate the selection and activation of its target genes during retinal development. Novel protein partners for Bm3b will be isolated using a yeast two-hybrid screening approach. Their functional relevance will be investigated by studying their effects on DNA-binding and transcriptional properties of Brn3b, and by examining their expression patterns and effects on Brn3b-mediated retinal ganglion cell differentiation; iii) to study in vivo the role of Barhl2 gene during retinal development. Two complementary approaches will be employed to analyze the in vivo functions of Barhl2 during retinogenesis. One is a loss-of-function approach involving targeted gene disruption to produce mice deficient for Barhl2. The other is a "gain-of-function" approach involving retrovirus-mediated overexpression of Barhl2 in the chick and mouse retina. Together, these proposed studies will provide important insights into the molecular regulatory networks that govern mammalian retinal development and may provide the foundation for better understanding and treatment of certain retinal disorders.